Caustic stable chromatography ligands

ABSTRACT

The present invention relates to chromatography ligands having improved caustic stability, e.g., ligands based on immunoglobulin-binding proteins such as,  Staphylococcal  protein A, as well as methods of making and using such ligands.

RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.Provisional Patent Application No. 61/203,664, filing date Dec. 24,2008, the entire content of which is incorporated herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to chromatography ligands having improvedcaustic stability, e.g., ligands based on immunoglobulin-bindingproteins such as, Staphylococcal protein A, as well as chromatographymatrices comprising such ligands.

BACKGROUND

Ligands used in affinity chromatography typically confer a highselectivity for the target molecule, thereby resulting in high yield andfast and economical purification of target molecules. Staphylococcalprotein A (SpA) based reagents and chromatography matrices have found awidespread use in the field of affinity chromatography for capture andpurification of antibodies as well as in antibody detection methods dueto its ability to bind IgG without significantly affecting the affinityof the immunoglobulin for antigen.

Accordingly, various reagents and media comprising protein A-ligandshave been developed and are commercially available including, forexample, ProSep®-vA High Capacity, ProSep® vA Ultra and ProSep®UltraPlus (Millipore) and Protein A Sepharose™, MabSelect™, MabSelectXtra™ and MabSelect SuRe® (GE Healthcare) and Poros MabCapture A™(Applied Biosystems).

In order to maintain selectivity and binding capacity of thechromatography ligands including, e.g., resins including SpA basedchromatography ligands, the ligand bound resins, referred to aschromatography matrices, have to be cleaned and are typically cleanedunder alkaline conditions, e.g., with sodium hydroxide. For example, astandard process which is used for cleaning and restoring the matrix isa cleaning-in-place (CIP) alkaline protocol, which typically involvestreatment of the matrix with 1M NaOH, pH 14. However, such harshtreatment is often undesirable, especially, where the ligand is aprotein or a protein-based molecule.

SUMMARY OF THE INVENTION

The present invention provides alkaline-stable SpA-based chromatographyligands which, for example, are capable of withstanding repeatedcleaning-in-place (CIP) cycles. More specifically, ligands according tothe invention are able to withstand conventional alkaline cleaning for aprolonged period of time, which renders the ligands attractivecandidates, especially for cost-effective large-scale purification ofimmunoglobulins.

In one aspect of the present invention, an alkaline-stablechromatography ligand comprises two or more domains of SpA. For example,in some embodiments according to this aspect, an alkaline-stablechromatography ligand is provided, which comprises two or more B domainsor two or more Z domains of Staphylococcus protein A (SpA), or afunctional fragment or variant thereof, where the two or more B domainsor two or more Z domains are attached to a chromatography resin at morethan one site on the resin.

In some embodiments according to this aspect, the ligand comprises threeor more B domains or three or more Z domains of SpA, or a functionalfragment or variant thereof, where the three or more B domains or threeor more Z domains are attached to a chromatography resin at more thanone site on the resin. In some other embodiments, the ligand comprisesfour or more B domains or four or more Z domains of SpA, where the fouror more B domains or four or more Z domains are attached to achromatography resin at more than one site on the resin. In yet otherembodiments, the ligand comprises five or more B domains or five or moreZ domains of SpA; or six or more B domains or six or more Z domains ofSpA; or seven or more B domains or seven or more Z domains of SpA, wherethe five or more B domains, or five or more Z domains, or six or more Bdomains, or six or more Z domains, or seven or more B domains or sevenor more Z domains are attached to a chromatography resin at more thanone site on the resin.

In another aspect according to the present invention, an alkaline-stablechromatography ligand comprises one or more isolated E, D, A, B, C or Zdomains of Staphylococcus protein A, where the one or more isolateddomains comprise one or more amino acid residues at position n+1 mutatedto any naturally occurring amino acid except cysteine (C), serine (S),alanine (A), glycine (G), asparagine (N), or glutamine (Q). In someembodiments, n represents the asparagine residue at position 23 of anisolated SpA domain. Exemplary ligands having a mutation at position 24of an isolated SpA domain where n represents an asparagine are shown inTable I.

TABLE I SpA domains including modifications Designation E; D, A, B, C orZ domain-glutamic acid 24 or aspartic E-D24M acid 24 replaced withmethionine D-E24M A-E24M B-E24M C-E24M Z-E24M E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24I acid 24 replaced withisoleucine D-E24I A-E24I B-E24I C-E24I Z-E24I E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24F acid 24 replaced withphenylalanine D-E24F A-E24F B-E24F C-E24F Z-E24F E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24T acid 24 replaced withthreonine D-E24T A-E24T B-E24T C-E24T Z-E24T E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24P acid 24 replaced with prolineD-E24P A-E24P B-E24P C-E24P Z-E24P E, D, A, B, C or Z domain-glutamicacid 24 or aspartic E-D24W acid 24 replaced with tryptophan D-E24WA-E24W B-E24W C-E24W Z-E24W E, D, A, B, C or Z domain-glutamic acid 24or aspartic E-D24R acid 24 replaced with arginine D-E24R A-E24R B-E24RC-E24R Z-E24R E, D, A, B, C or Z domain-glutamic acid 24 or asparticE-D24V acid 24 replaced with valine D-E24V A-E24V B-E24V C-E24V Z-E24VE, D, A, B, C or Z domain-glutamic acid 24 or aspartic E-D24L acid 24replaced with leucine D-E24L A-E24L B-E24L C-E24L Z-E24L E, D, A, B, Cor Z domain-glutamic acid 24 or aspartic E-D24Y acid 24 replaced withtyrosine D-E24Y A-E24Y B-E24Y C-E24Y Z-E24Y E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24H acid 24 replaced withhistidine D-E24H A-E24H B-E24H C-E24H Z-E24H E, D, A, B, C or Zdomain-glutamic acid 24 or aspartic E-D24K acid 24 replaced with lysineD-E24K A-E24K B-E24K C-E24K Z-E24K E, D, A, B, C or Z domain-glutamicacid 24 or aspartic D-E24D acid 24 replaced with glutamic acid A-E24DB-E24D C-E24D Z-E24D

The single letter codes for the naturally occurring amino acids as wellas the corresponding three letter codons encoding for each amino acidare depicted in Table II. In general, due to the degeneracy of thecodon, more than one three letter codon can encode for the same aminoacid.

TABLE II T C A G T TTT Phe(F) TCT Ser(S) TAT Tyr(Y) TGT Cys(C)TTC Phe(F) TCC Ser(S) TAC TGC TTA Leu(L) TCA Ser(S) TAA Stop TGA StopTTG Leu(L) TCG Ser(S) TAG Stop TGG Trp(W) C CTT Leu(L) CCT Pro(P)CAT His(H) CGT Arg(R) CTC Leu(L) CCC Pro(P) CAC His(H) CGC Arg(R)CTA Leu(L) CCA Pro(P) CAA Gln(Q) CGA Arg(R) CTG Leu(L) CCG Pro(P)CAG Gln(Q) CGG Arg(R) A ATT Ile(I) ACT Thr(T) AAT Asn(N) AGT Ser(S)ATC Ile(I) ACC Thr(T) AAC Asn(N) AGC Ser(S) ATA Ile(I) ACA Thr(T)AAA Lys(K) AGA Arg(R) ATG Met(M) ACG Thr(T) AAG Lys(K) AGG Arg(R) GGTT Val(V) GCT Ala(A) GAT Asp(D) GGT Gly(G) GTC Val(V) GCC Ala(A)GAC Asp(D) GGC Gly(G) GTA Val(V) GCA Ala(A) GAA Glu(E) GGA Gly(G)GTG Val(V) GCG Ala(A) GAG Glu(E) GGG Gly(G)

Also encompassed by the present invention is a chromatography matrixcomprising a ligand according to one or more aspects of the inventioncoupled to a solid support such as, e.g., at least one insolublecarrier.

Additionally, provided herein are methods of using the ligands describedherein. Accordingly, a method of affinity purifying one or more targetmolecules (e.g., immunoglobulins) from a sample is provided, where themethod comprising the steps of: (a) providing a sample comprising one ormore target molecules (e.g., immunoglobulins); (b) contacting the samplewith a matrix according to the invention under conditions such that theone or more target molecules (e.g., immunoglobulins) bind to the matrix;and (c) recovering the one or more bound target molecules (e.g.,immunoglobulins) by eluting under suitable conditions such as, forexample, a suitable pH.

In some embodiments, an alkaline-stable chromatography ligand accordingto the present invention retains at least 95% of its binding capacityafter 5 hours, or after 10 hours, after 15 hours, or after 20 hours, orafter 25 hours, or after 30 hours of incubation in 0.5 M NaOH.

The immunoglobulins which are capable of being bound by the variousligands described herein include, e.g., IgG, IgA and IgM, or any fusionprotein comprising antibody and any fragment of antibody.

Also provided herein are nucleic acid molecules encoding the variousligands described herein, as well as host cells including such nucleicacid molecules. In some embodiments, a host cell is a prokaryotic cell.In other embodiments, a host cell is a eukaryotic cell.

Additionally, the present invention encompasses a library ofpolypeptides comprising one or more ligands described herein, andfunctional fragments and variants thereof. In yet another embodiment,the present invention provides a library of nucleic acid moleculesencoding one or more ligands encompassed by the present invention orencoding functional fragments and variants thereof.

In some embodiments, the present invention provides SpA based ligandswhich exhibit altered (increased or decreased) binding to a Fab portionof an immunoglobulin compared to the previously known SpA ligands, whileretaining the ability to bind the Fc portion of the immunoglobulin. Inone embodiment, an SpA based ligand according to the present inventionexhibits decreased binding to a Fab portion of an immunoglobulincompared to wild type SpA. In an exemplary embodiment, analkaline-stable chromatography ligand according to the present inventionfurther includes the amino acid glycine at position 29 replaced with analanine. In another embodiment, an alkaline stable chromatography ligandaccording to the present invention further includes the glycine atposition 29 replaced with an amino acid other than alanine ortryptophan.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid sequences for the wild type (wt) IgGbinding domains of SpA, represented by SEQ ID NOs:1-5. SEQ ID NO:1represents the nucleic acid sequence for the wt E domain; SEQ ID NO: 2represents the nucleic acid sequence for the wt D domain; SEQ ID NO: 3represents the nucleic acid sequence for the wt A domain; SEQ ID NO: 4represents the nucleic acid sequence for the wt B domain; and SEQ ID NO:5 represents the nucleic acid sequence for the wt C domain.

FIG. 2 depicts the nucleic acid sequence for the Z domain of SpA,represented by SEQ ID NO: 6.

FIG. 3 depicts the amino acid sequence alignments of the wild type (wt)IgG binding domains of SpA (E, D, A, B and C). SEQ ID NO: 7 representsthe amino acid sequence of the wt E domain; SEQ ID NO: 8 represents theamino acid sequence of the wt D domain; SEQ ID NO: 9 represents theamino acid sequence of the wt A domain; SEQ ID NO: 10 represents theamino acid sequence of the wt B domain; and SEQ ID NO: 11 represents theamino acid sequence of the wt C domain.

FIG. 4 depicts the amino acid sequence of the Z domain, represented bySEQ ID NO: 12.

FIG. 5 depicts a schematic of the plasmid pJ56:8620. The sequence of theHis6 tag is disclosed in SEQ ID NO:42.

FIG. 6 depicts the amino acid sequences for the his-tagged wt B domainof SpA as well as the various his-tagged n+1 mutants. SEQ ID NO: 13represents the amino acid sequence for the his-tagged wt B domain of SpAand SEQ ID NOs: 14, 15; 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26represent the amino acid sequences for the n+1 B domain mutants E24M,E24I, E24F, E24T, E24P, E24W, E24R, E24V, E24L, E24Y, E24H, E24K. andE24D, respectively. The sequence of the His6 tag is disclosed in SEQ IDNO:42.

FIG. 7 depicts the nucleic acid sequences for the his-tagged wt B domainof SpA as well as nucleic acid sequences encoding the various his-taggedn+1 mutants. SEQ ID NO: 27 represents the nucleic acid sequence encodingthe his-tagged wt B domain of SpA and SEQ ID NOs: 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39 and 40 represent the nucleic acid sequencesencoding the n+1 B domain mutants E24M, E24I, E24F, E24T, E24P, E24W,E24R, E24V, E24L, E24Y, E24H, E24K and E24D, respectively. The sequenceof the His6 tag is disclosed in SEQ ID NO:42.

FIG. 8 depicts a bar graph summarizing the results of an exemplaryexperiment to assay for residual IgG binding in the various his-taggedn+1 B domain mutants, where n represents the asparagine at position 23,using the high-throughput ELISA based assay described herein. The X-axisrepresents various n+1 B domain mutants having a mutation at position 24and the Y-axis represents the percent IgG binding remaining after a sixhour exposure to 1N NaOH. As shown in the graph, n+1 B domain mutantshaving amino acids which contain bulky side chains at position 24exhibit increased caustic stability relative to the wt B domain.

FIG. 9 depicts the results of an exemplary experiment to assay for thepercent retained IgG binding capacity of the various SpA domain trimers(i.e., EEE, DDD, AAA, BBB, CCC and ZZZ) subsequent to their multipointattachment to an agarose resin. nPrA refers to the wt SpA. The X-axisrepresents the number of cycles of caustic exposure, where each cycleconsists of a 15 minute exposure to 0.5 N NaOH. The Y-axis representsthe retained IgG binding capacity of the attached SpA ligands. As shownin the graph in FIG. 9, the ligands including C or Z domain trimers areroughly equivalent in their caustic stability and are more causticstable than the ligands including B domain trimers, which are morecaustic stable than the ligands including A domain trimers, which aremore caustic stable than the ligands including E or D domain trimers.

FIG. 10 depicts the results of an exemplary experiment to assay for thepercent retained IgG binding capacity of attached ligands according tothe invention, containing three (B3), or four (B4), or five (B5) Bdomains, attached via multipoint attachment to an agarose resin. Also,attached ligands containing five B domains or seven B domains andadditionally including a mutation to reduce Fab binding (G29A) are used,referred to as B5-NF and B7-NF, respectively. nPrA refers to the wt SpA.The X-axis represents the number of cycles of caustic exposure, whereeach cycle consists of a 15 minute exposure to 0.5 N NaOH. The Y-axisrepresents the retained IgG binding capacity of the attached SpAligands. As shown in the graph in FIG. 10, the level or extent ofcaustic stability is directly proportional to the number of B domains inthe ligands and is not altered by the G29A mutation to decrease Fabbinding.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides SpA based alkaline-stable chromatographyligands, and in particular, ligands based on one or more domains of SpA.Previously described exemplary SpA based alkaline-stable chromatographyligands include, for example, those described in International PCTpatent application no. WO2008/039141, which discusses alkaline-stablechromatography ligands based on the C domain of SpA which are capable ofbinding the Fab portions of antibodies and are coupled to an insolublecarrier at a single site using a terminal coupling group; and thosedescribed in U.S. Pat. No. 6,831,161, which discusses SpA based alkalinebased chromatography ligands where one or more asparagine amino acidresidues have modified.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

As used herein, the term “SpA” or “protein A of Staphylococcus aureus,”refers to a 42 Kda multi-domain protein isolated from the bacteriumStaphylococcus aureus. SpA is bound to the bacterial cell wall via itscarboxy-terminal cell wall binding region, referred to as the X domain.At the amino-terminal region, it includes five immunoglobulin-bindingdomains, referred to as E, D, A, B, and C (Sjodhal, Eur J Biochem.September 78(2):471-90 (1977); Uhlen et al., J Biol Chem. February259(3):1695-702 (1984). Each of these domains contains approximately 58amino acid residues, and they share 65-90% amino acid sequence identity.The Z domain of SpA is an engineered analogue of the B domain of SpA andincludes an alanine instead of a glycine residue at position 29(Nilsson, et al., Protein engineering, Vol. 1, No. 2, 107-113; 1987.).Each of the E, D, A, B and C domains of SpA possess distinct Ig-bindingsites. One site is for Fcγ (the constant region of IgG class of Ig) andthe other is for the Fab portion of certain Ig molecules (the portion ofthe Ig that is responsible for antigen recognition). It has beenreported that each of the domains contains a Fab binding site. Thenon-Ig binding portion of SpA is located at the C-terminus and isdesignated the X region or X-domain.

The cloning of the gene encoding SpA is described in U.S. Pat. No.5,151,350, the entire contents of which are incorporated by referenceherein in their entirety.

The present invention provides SpA-based alkaline-stable chromatographyligands. In some aspects according to the present invention, an alkalinestable chromatography ligand comprises two or more, or three or more, orfour or more, or five or more, or six or more, or seven or more ofisolated wt B or Z domain of SpA. In other aspects according to thepresent invention, an alkaline stable chromatography ligand comprisesone or more isolated E, D, A, B, C or Z domains of SpA, where the one ormore isolated domains comprise one or more amino acid residues atposition n+1 mutated to an amino acid selected from the group consistingof tryptophan, arginine, threonine, isoleucine, valine and methionine,wherein n represents an asparagine.

In a particular embodiment, the present invention provides achromatography ligand comprising two or more B domains of SpA, attachedto a chromatography resin at more than one site on the resin. In anotherembodiment, the present invention provides a chromatography ligandcomprising two or more Z domains of SpA, attached to a chromatographyresin at more than one site on the resin. In yet another embodiment, achromatography ligand comprises two or more C domains of SpA, attachedto a chromatography resin at more than one site on the resin.

Also encompassed by the present invention are amino acid variants ofSpA, which may differ from the parent amino acid sequence from whichthey are derived, in the substitution, deletion and/or insertion of oneor more amino acids anywhere within the parent amino acid sequence andare alkaline stable. In some embodiments, amino acid sequence variantswill possess at least about 70%, or at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98% identity withthe parent sequence (i.e. wt SpA domains or Z domain), where suchvariants are alkaline stable. In a particular embodiment, variants ofSpA further include the glycine amino acid residue at position 29replaced by an amino acid residue other than alanine or tryptophan,while retaining its alkaline stability.

The term “functional variant” of a protein means herein a variantprotein, where the function, in relation to the invention defined asalkaline stability, is essentially retained. Functional variantsinclude, and are not limited to, SpA variants including more than domainof SpA, e.g., dimers, trimers, multimers of various domains of SpA andSpA variants having a deletion, substitution and/or addition of one ormore amino acids in one or more wild type domains of SpA, whileretaining the alkaline stability, as defined herein.

The term “parental molecule” is used herein for the correspondingprotein in the form before it is modified according to the invention ora mutation according to the invention has been introduced.

The term “sequence identity” means that two nucleotide or amino acidsequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 70% sequence identity,or at least 80% sequence identity, or at least 85% sequence identity, orat least 90% sequence identity, or at least 95% sequence identity ormore. For sequence comparison, typically one sequence acts as areference sequence (e.g., parent sequence), to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

As used interchangeably herein, the terms “E domain,” “E domain of SpA,”and “E domain of Staphylococcus protein A,” refer to the polypeptidewhose amino acid sequence is set forth in SEQ ID NO:7 or that encodedby, e.g., the nucleotide sequence set forth in SEQ ID NO: 1. The “Edomain” is a 51 amino acid polypeptide that folds into a three-helixbundle structure. It is capable of binding Fc via residues on thesurface of helices 1 and 2, or to Fab via residues on the surface ofhelices 2 and 3. In some embodiments, an E domain according to theinvention is at least 70% identical, or at least 80% identical, or atleast 90% identical or at least 95% or more identical in sequence to theamino acid sequence set forth in SEQ ID NO:7.

As used interchangeably herein, the terms “D domain,” “D domain of SpA,”and “D domain of Staphylococcus protein A,” refer to the polypeptidewhose amino acid sequence is set forth in SEQ ID NO: 8 or that encodedby e.g., the nucleotide sequence set forth in SEQ ID NO: 2. The “Ddomain” is a 61 amino acid polypeptide that folds into a three-helixbundle structure. It is capable of Fc binding via residues on thesurface of helices 1 and 2, or to Fab via residues on the surface ofhelices 2 and 3. In some embodiments, a D domain according to theinvention is at least 70% identical, or at least 80% identical, or atleast 90% identical or at least 95% or more identical in sequence to theamino acid sequence set forth in SEQ ID NO: 8.

As used interchangeably herein, the terms “A domain,” “A domain of SpA,”and “A domain of Staphylococcus protein A,” refer to the polypeptidewhose amino acid sequence is set forth in SEQ ID NO: 3 or that encodedby, e.g., the nucleotide sequence set forth in SEQ ID NO: 9. The “Adomain” is a 58 amino acid polypeptide that folds into a three-helixbundle structure. It is capable of Fc binding via residues on thesurface of helices 1 and 2, or to Fab via residues on the surface ofhelices 2 and 3. In some embodiments, an A domain according to theinvention is at least 70% identical, or at least 80% identical, or atleast 90% identical or at least 95% or more identical in sequence to theamino acid sequence set forth in SEQ ID NO: 3.

As used interchangeably herein, the terms “B domain,” “B domain of SpA,”and “B domain of Staphylococcus protein A,” refer to the polypeptidewhose amino acid sequence is set forth in SEQ ID NO: 10 or that encodedby, e.g., the nucleotide sequence set forth in SEQ ID NO: 4. The “Bdomain” is a 58 amino acid polypeptide that folds into a three-helixbundle structure. It is capable of Fc binding via residues on thesurface of helices 1 and 2, or to Fab via residues on the surface ofhelices 2 and 3. In some embodiments, a B domain according to theinvention is at least 70% identical, or at least 80% identical, or atleast 90% identical or at least 95% or more identical in sequence to theamino acid sequence set forth in SEQ ID NO: 10.

As used interchangeably herein, the terms “C domain,” “C domain of SpA,”and “C domain of Staphylococcus protein A,” refer to the polypeptidewhose amino acid sequence is set forth in SEQ ID NO: 11 or that encodedby, e.g., the nucleotide sequence set forth in SEQ ID NO: 5. The “Cdomain” is a 58 amino acid polypeptide that folds into a three-helixbundle structure. It is capable of Fc binding via residues on thesurface of helices 1 and 2, or to Fab via residues on the surface ofhelices 2 and 3. In some embodiments, a C domain according to theinvention is at least 70% identical, or at least 80% identical, or atleast 90% identical or at least 95% or more identical in sequence to theamino acid sequence set forth in SEQ ID NO: 11.

As used interchangeably herein, the terms “Z domain,” “Z domain of SpA”and “Z domain of protein A,” refer to the three helix, 59 amino acidpolypeptide that is a variant of the B domain of protein A. The aminoacid sequence of the Z domain is set forth in SEQ ID NO: 12. Anexemplary Z domain is described in Nilsson et al., Protein Engng.,1:107-113 (1997), the entire contents of which are incorporated byreference herein.

The term “alkaline-stable,” “alkaline stability,” “caustic stable” or“caustic stability,” as used herein, generally refers to the ability ofa chromatography ligand according to the present invention, either aloneor when immobilized onto a chromatography resin, to withstand repeatedcleaning-in-place (CIP) cycles using alkaline wash without losing itsbinding capacity. In general, it is assumed that a resin, by itself,onto which a ligand according to the invention is immobilized,contributes to less than a 5% change in stability after having beensoaked in 0.5 M NaOH for up to 30 hours. For example, in someembodiments, chromatography ligands according to the invention are ableto withstand conventional alkaline cleaning for a prolonged period oftime, which renders the ligands attractive candidates, especially forcost-effective large-scale purification of immunoglobulins. In someembodiments, a ligand according to the present invention exhibits animproved chemical stability in an alkaline environment, which may bedefined, for example, as that having an increased pH value such as aboveabout 10, or up to about 13 or 14. Alternatively, the alkalineenvironment can be defined by the concentration of a base, e.g., about1.0 M NaOH, or about 0.7 M NaOH, or about 0.5 M NaOH. In one embodiment,alkaline stability refers to the ability of an alkaline-stablechromatography ligand according to the present invention to retain atleast 80%, or at least 85%, or at least 90%, or at least 95% of itsbinding capacity after 5 hours, or after 10 hours, after 15 hours, orafter 20 hours, or after 25 hours, or after 30 hours of incubation in0.5 M NaOH. In another embodiment, alkaline stability refers to adecrease in the binding capacity of the ligand by less than 70%, or lessthan 60%, or less than 50%, or less than 30% even after treatment with0.5 M NaOH for 5 hours or 7.5 hours or 10 hours or 15 hours or 20 hoursor 25 hours or 30 hours.

In some embodiments, SpA based chromatography ligands according to thepresent invention exhibit an increased or improved alkaline stability ascompared to wild type SpA.

Alkaline stability can be readily measured by one of ordinary skill inthe art using routine experimentation and/or as described herein.

The term “chromatography,” as used herein, refers to a dynamicseparation technique which separates the analyte of interest (e.g., animmunoglobulin) from other molecules in the mixture and allows it to beisolated. Typically, in a chromatography method, a mobile phase (liquidor gas) transports a sample containing the analyte of interest across orthrough a stationary phase (normally solid) medium. Differences inpartition or affinity to the stationary phase separate differentanalytes while mobile phase carries the different analytes out atdifferent time.

The term “affinity chromatography,” as used herein, refers to a mode ofchromatography where the analyte to be separated is isolated by itsinteraction with a molecule (e.g., an alkaline stable chromatographyligand) which specifically interacts with the analyte. In oneembodiment, affinity chromatography involves the addition of a samplecontaining a target analyte (e.g., an immunoglobulin) to a solid supportwhich carries on it an alkaline stable chromatography ligand, asdescribed herein.

The term “protein A affinity chromatography,” as used herein, refers tothe separation or isolation of substances using protein A or SpAligands, such as those described herein, where the SpA or protein Aligand is immobilized, e.g., on a solid support. Examples of protein Aaffinity chromatography media/resin known in the art include thosehaving the protein A immobilized onto a controlled pore glass backbone,e.g., PROSEP A™ and PROSEP vA™ media/resin (Millipore); those havingprotein A immobilized onto a polystyrene solid phase, e.g., the POROS50A™ and Poros MabCapture A™ media/resin (Applied Biosystems, Inc.); andthose having protein A immobilized on an agarose solid support, e.g.,rPROTEIN A SEPHAROSE FAST FLOW™ or MABSELECT™ columns (AmershamBiosciences).

The term “immunoglobulin,” “Ig” or “antibody” (used interchangeablyherein) refers to a protein having a basic four-polypeptide chainstructure consisting of two heavy and two light chains, said chainsbeing stabilized, for example, by interchain disulfide bonds, which hasthe ability to specifically bind antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind antigen. The term “domain” refers to a globular region of a heavyor light chain polypeptide comprising peptide loops (e.g., comprising 3to 4 peptide loops) stabilized, for example, by β-pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of antibodylight chains are referred to interchangeably as “light chain constantregions”, “light chain constant domains”, “CL” regions or “CL” domains.The “constant” domains of antibody heavy chains are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains. The “variable” domains ofantibody light chains are referred to interchangeably as “light chainvariable regions”, “light chain variable domains”, “VL” regions or “VL”domains. The “variable” domains of antibody heavy chains are referred tointerchangeably as “heavy chain variable regions”, “heavy chain variabledomains”, “VH” regions or “VH” domains.

Immunoglobulins or antibodies may be monoclonal or polyclonal and mayexist in monomeric or polymeric form, for example, IgM antibodies whichexist in pentameric form and/or IgA antibodies which exist in monomeric,dimeric or multimeric form. The term “fragment” refers to a part orportion of an antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody or antibody chain.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. Exemplary fragments include Fab, Fab′,F(ab′)2, Fc and/or Fv fragments.

The term “antigen-binding fragment” refers to a polypeptide portion ofan immunoglobulin or antibody that binds an antigen or competes withintact antibody (i.e., with the intact antibody from which they werederived) for antigen binding (i.e., specific binding). Binding fragmentscan be produced by recombinant DNA techniques, or by enzymatic orchemical cleavage of intact immunoglobulins. Binding fragments includeFab, Fab′, F(ab′)₂, Fv, single chains, and single-chain antibodies.

Also encompassed are fusion proteins including an antibody or fragmentthereof as a part of the fusion protein.

The terms “polynucleotide” and “nucleic acid molecule,” usedinterchangeably herein, refer to polymeric forms of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides. These termsinclude a single-, double- or triple-stranded DNA, genomic DNA, cDNA,RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically or biochemically modified,non-natural or derivatized nucleotide bases. The backbone of thepolynucleotide can comprise sugars and phosphate groups (as maytypically be found in RNA or DNA), or modified or substituted sugar orphosphate groups. In addition, a double-stranded polynucleotide can beobtained from the single stranded polynucleotide product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. A nucleic acid molecule can take many different forms, e.g., agene or gene fragment, one or more exons, one or more introns, mRNA,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. As used herein, “DNA” or “nucleotidesequence” includes not only bases A, T, C, and G, but also includes anyof their analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides. In a particular embodiment, anucleic acid molecule comprises a nucleotide sequence encoding a variantof SpA.

The term “Fc-binding,” “binds to an Fc portion” or “binding to an Fcportion” refers to the ability of an alkaline stable chromatographyligand described herein, to bind to the crystallizable part (Fc) of anantibody. In some embodiments, ligand according to the present inventionbinds an Fc portion of an antibody (e.g., human IgG1, IgG2 or IgG4) withan affinity of at least 10⁻⁷ M, or at least 10⁻⁸ M, or at least 10⁻⁹ M.

As used herein, the term “Fab binding” or “binding to a Fab portion”refers to the ability of an alkaline stable chromatography liganddescribed herein, to bind to a Fab region of an antibody or animmunoglobulin molecule. The term “reduced binding to a Fab portion”refers to any decrease in binding to a Fab (or F(ab)₂) portion of animmunoglobulin molecule by an SpA based ligand according to the presentinvention relative to the wt SpA, where the ligand further includes amutation in one or more amino acids. In an exemplary embodiment, aligand according to the present invention further includes the glycineresidue at position 29 replaced with an alanine. In another embodiment,a ligand according to the present invention further includes the glycineat position 29 replaced with an amino acid other than alanine ortryptophan. In one embodiment, binding to a Fab portion of animmunoglobulin molecule is undetectable using conventional techniques inthe art and those described herein. Binding to an immunoglobulinmolecule can be detected using well known techniques including thosedescribed herein and including but not limited to, for example, affinitychromatography and Surface Plasmon Resonance Analysis. In someembodiments, an immunoglobulin binding protein encompassed by thepresent invention binds an immunoglobulin molecule with an affinity ofat least 10⁻¹⁰M.

II. Generation of SpA Based Molecules for Use as Chromatography Ligands

The SpA based chromatography ligands encompassed by the presentinvention can be made using any suitable methods known in the art.

For example, as an initial step, standard genetic engineeringtechniques, e.g., those described in the laboratory manual entitledMolecular Cloning by Sambrook, Fritsch and Maniatis, may be used for thegeneration of nucleic acids which express the SpA ligand moleculesdescribed herein.

In some embodiments, a nucleic acid molecule encoding one or moredomains of SpA or portions thereof can be cloned into a suitable vectorfor expression in an appropriate host cell. Suitable expression vectorsare well known in the art and typically include the necessary elementsfor the transcription and translation of the variant SpA codingsequence.

SpA molecules described herein may also be synthesized chemically fromamino acid precursors for fragments using methods well known in the art,including solid phase peptide synthetic methods such as the Boc(tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxy carbonyl)approaches (see, e.g., U.S. Pat. Nos. 6,060,596; 4,879,378; 5,198,531;5,240,680).

Expression of SpA molecules described herein can be accomplished incells from eukaryotic hosts such as yeasts, insects or mammals, or inprokaryotic host cells, e.g., bacteria such as E. coli.

In some embodiments, SpA molecules or fragments and variants thereof maybe expressed on the surface of a bacteriophage such that each phagecontains a DNA sequence that codes for an individual SpA moleculedisplayed on the phage surface. The affinity of the SpA molecule for animmunoglobulin can be readily assayed for using standard techniques inthe art and those described herein, e.g., ELISA and Biacore™ 2000standard set-up (Biacore AB, Uppsala Sweden). It is desirable that thebinding affinity of an SpA molecule of the present invention to animmunoglobulin is at least comparable with that of the parent molecule.Furthermore, it is desirable that the alkaline stability of the SpAmolecule is generally improved over that of the parent molecule.

III. Assaying for Alkaline Stability of the SpA Molecules

Subsequent to the generation and purification of a suitable SpA ligandmolecule, as described herein, the alkaline stability of the moleculecan be assayed using standard techniques in the art and those describedherein. For example, the alkaline stability of an SpA molecule accordingto the invention can assayed using routine treatment with NaOH at aconcentration of about 0.5M, e.g., as described in the experimental partbelow.

In some embodiments, alkaline stable SpA molecules exhibit an“increased” or “improved” alkaline stability, meaning that the moleculesare stable under alkaline conditions for an extended period of timerelative to wild type SpA. Previously, it has been reported that SpAmolecules based on the wild type C domain of SpA or having a mutation ofone or more asparagine residues provides an improved chemical stabilityand hence a decreased degradation rate in environments wherein the pH isabove about 10, such as up to about 13 or 14.

The present invention is based on the surprising and unexpecteddiscovery of novel SpA molecules which exhibit increased alkalinestability even when they are based on domains other than the C domain orhave mutations in amino acids other than asparagines. For example, thepresent invention provides B or Z domain based alkaline stable SpAmolecules and SpA molecules which have a mutation in an amino acid atposition n+1, where n represents an asparagine (e.g., asparagine atposition 23).

In some embodiments, subsequent to the generation of the SpA ligandsaccording to the present invention, alkaline stability of the ligands isevaluated using a novel high throughput immunological assay, describedin more detail in the Examples infra. The assay is based on theassumption that degradation of SpA in response to extended causticexposure is reflected as a loss or reduction in IgG binding. Briefly,soluble SpA based ligands are treated for about 6 hours with eitherwater or 1.0M NaOH. Hydrophobic interactions are used to attachmicrogram quantities of neutralized candidate ligands to a solid supportin the form of an ELISA plate, e.g., a 96 well plate. IgG binding isthen assessed for each candidate ligand before and after exposure to1.0M NaOH. Enhanced caustic stability is indicated when the amount ofresidual IgG binding to a ligand following caustic exposure exceeds thatof wild type SpA or parental SpA from which it is derived.

IV. Supports Used for the Preparation of Chromatography Matrices

In some embodiments, alkaline stable SpA ligands encompassed by thepresent invention are attached to a support, e.g., a solid support or asoluble support, to generate a chromatography matrix suitable for theseparation of biomolecules such as, e.g., immunoglobulins.

In some embodiments, a ligand according to the present invention isattached to a solid support. Without wishing to be bound by theory, itis contemplated that any suitable solid support may be used for theattachment of a ligand according to the invention. For example, solidsupport matrices include, but are not limited to, controlled pore glass,silica, zirconium oxide, agarose, polymethacrylate, polyacrylate,polyacrylamide, and polystyrene.

It is contemplated that any porous material that contributes to lessthan a 5% change in alkaline stability of the attached ligand aftersoaking for about 30 hours in 0.5 M NaOH may be used as a solid support.

A porous material used as a solid support may be comprised of ahydrophilic compound, a hydrophobic compound, an oleophobic compound, anoleophilic compound or any combination thereof. The porous material maybe comprised of a polymer or a copolymer. Examples of suitable porous,materials, include, but are not limited to polyether sulfone, polyamide,e.g., nylon, polysaccharides such as, for example, agarose andcellulose, polyacrylate, polymethacrylate, polyacrylamide,polymethacrylamide, polytetrafluoroethylene, polysulfone, polyester,polyvinylidene fluoride, polypropylene, polyethylene, polycarbonate, afluorocarbon, e.g. poly (tetrafluoroethylene-co-perfluoro(alkyl vinylether)), glass, silica, zirconia, titania, ceramic, and metal.

The porous material may be comprised of an organic or inorganicmolecules or a combination of organic and inorganic molecules and may becomprised of one or more functional groups, e.g., a hydroxyl group, athiol group, an amino group, a carbonyl group, or a carboxylic acidgroup, suitable for reacting, e.g., forming covalent bonds for furtherchemical modification in order to covalently bond to a protein. Inanother embodiment, the porous material may not possess a functionalgroup but can be coated with a layer of material that bears functionalgroups such as, an hydroxyl group, a thiol group, an amino acid group, acarbonyl group, or a carboxylic acid group.

In some embodiments, a conventional affinity separation matrix is used,e.g., of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carbonyl (—CHO, or RCO—R′), carboxamido (—CONH₂, possibly inN-substituted forms), amino (—NH₂, possibly in substituted form), oligo-or polyethylenoxy groups on their external and, if present, also oninternal surfaces. In one embodiment, the polymers may, for instance, bebased on polysaccharides, such as dextran, starch, cellulose, pullulan,agarose etc, which advantageously have been cross-linked, for instancewith bisepoxides, epihalohydrins, 1,2,3-trihalo substituted lowerhydrocarbons, to provide a suitable porosity and rigidity. In anotherembodiment, the solid support comprises porous agarose beads. Thevarious supports used in the present invention can be readily preparedaccording to standard methods known in the art, such as, for example,inverse suspension gelation described, e.g., in Hjerten, Biochim BiophysActa 79(2), 393-398 (1964). Alternatively, the base matrices can becommercially available products, such as Sepharose™ FastFlow (GEHealthcare, Uppsala, Sweden).

In some embodiments, especially advantageous for large-scaleseparations, the support is adapted to increase its rigidity, and hencerenders the matrix more suitable for high flow rates.

Alternatively, the solid support can be based on synthetic polymers,such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers can be easily producedaccording to standard methods, see e.g., Arshady, Chimica e L'Industria70(9), 70-75 (1988). Alternatively, a commercially available product,such as Source™ (GE Healthcare, Uppsala, Sweden) and Poros (AppliedBioSystems, Foster City, Calif.) may be used.

In yet other embodiments, the solid support comprises a support ofinorganic nature, e.g. silica, zirconium oxide etc. The surface ofinorganic matrices is often modified to include suitable reactive groupsfor further reaction to SpA and its variants. Examples include CMZirconia (Ciphergen-BioSepra (CergyPontoise, France) and CPG®(Millipore).

In some embodiments, the polymers may, for instance, be based onzirconia or silica or controlled pore glass, which may be modified toeither contain reactive groups and/or sustain caustic soaking, to becoupled to ligands.

Exemplary solid support formats include, but are not limited to, a bead,a gel, a membrane, a cassette, a column, a chip, a slide, a plate or amonolith.

With respect to the format of a matrix, in one embodiment, it is in theform of a porous monolith. In an alternative embodiment, the matrix isin beaded or particle form that can be porous or non-porous. Matrices inbeaded or particle form can be used as a packed bed or in a suspendedform. Suspended forms include those known as expanded beds and puresuspensions, in which the particles or beads are free to move. In caseof monoliths, packed bed and expanded beds, the separation procedurecommonly follows conventional chromatography with a concentrationgradient. In case of pure suspension, batch-wise mode will be used.Also, solid support in forms such as a surface, a chip, a capillary, ora filter may be used.

The matrix could also be in the form of membrane in a cartridge. Themembrane could be in flat sheet, spiral, or hollow fiber format.

In another embodiment, a ligand according to the present invention isattached to a soluble support, e.g., a soluble polymer. Exemplarysoluble supports include, but are not limited to, a bio-polymer such as,e.g., a protein or a nucleic acid. In some embodiments, biotin maybeused as a soluble polymer, e.g., as described in US Patent PublicationNo. 20080108053. For example, biotin may be bound to a ligand, e.g., anSpA based caustic stable ligand according to the present invention,which subsequent to being bound to the ligand can be used for isolatinga protein of interest, e.g., an antibody or fragment thereof, e.g.,present in a crude mixture and the protein of interest can be isolatedor separated via precipitation of the biotin-ligand-protein polymercomplex in either a reversible or irreversible fashion. The polymer mayalso be a synthetic soluble polymer, such as, for example, including butnot limited, to a polymer containing negatively charged groups(carboxylic or sulfonic), positively charged groups (quarternary amine,tertiary amine, secondary or primary groups), hydrophobic groups (phenylor butyl groups), hydrophilic groups (hydroxyl, or amino groups) or acombination of the above. Exemplary synthetic soluble polymers can befound in International PCT Publication No. WO2008091740 and U.S.Publication No. US20080255027, the entire teachings of each of which areincorporated by reference herein. These polymers, upon specific physicalchanges in one or more conditions such as pH, conductivity ortemperature, can be used to purify the protein of interest viaprecipitation in either a reversible or an irreversible fashion.Synthetic soluble polymers may be used alone or may be coupled with acaustic stable ligand according to the present invention and used forcapture/purification of a protein of interest such as, e.g., an antibodyor a fragment thereof, via precipitation in either a reversible or anirreversible fashion.

V. Methods for Attaching a Ligand to a Support

Any suitable technique may be used for attaching a ligand to a support,e.g., a solid support including those well known in the art anddescribed herein. For example, in some embodiments, the ligand may beattached to a support via conventional coupling techniques utilizing,e.g. amino and/or carboxy groups present in the ligand. For example,bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc. arewell-known coupling reagents. In some embodiments, a spacer isintroduced between the support and the ligand, which improves theavailability of the ligand and facilitates the chemical coupling of theligand to the support. Alternatively, the ligand may be attached to thesupport by non-covalent bonding, such as physical adsorption orbiospecific adsorption.

In various embodiments encompassed by the present invention, the ligandis attached to a solid support such as, for example, a chromatographyresin at more than one site, thereby resulting in a chromatographymatrix.

Attachment of an alkaline stable SpA based chromatography ligand to asolid support can be achieved via many different ways known, most ofwhich are well known in the art, as well as those described herein. See,e.g., Hermanson et al., Immobilized Affinity Ligand Techniques, AcademicPress, pp. 51-136 (1992).

For example, protein ligands can be coupled to a solid support viaactive groups on either the surface of the solid support or the proteinligand, such as, for example, hydrolxyl, thiol, epoxide, amino,carbonyl, epoxide, or carboxylic acid group. Attachment can be achievedusing known chemistries including, but not limited to, use of cyanogenbromide (CNBr), N-hydroxyl succinimide ester, epoxy (bisoxirane)activation, and reductive amination.

For example, thiol directed protein coupling has been described in theliterature. See, e.g., Ljungquist, et al. Eur. J. Biochem. Vol 186, pp.558-561 (1989). This technique has been previously applied for couplingSpA to a solid support. Since wild type SpA does not contain thiolgroups, the attachment is achieved by recombinantly inserting a thiolcontaining cysteine at the C-terminus of SpA. See, e.g., U.S. Pat. No.6,399,750. Several commercial products such as MabSelect™, MabSelect™Xtra and MabSelect™ SuRe are produced via this mechanism. It has beenreported that this terminal cysteine only reacts with the epoxide groupon the solid surface, thereby resulting in single point attachment ofthe SpA to the solid support. See, e.g., Process Scale Bioseparationsfor the Biopharmaceutical Industry, CRC Press, 2006, page 473.

In case of the present invention, in certain embodiments, SpA basedchromatography ligands comprising two or more domains of SpA areattached to a solid support at more than one site via non-discriminate,multipoint attachment. In general, SpA contains abundant free aminogroups from numerous lysines in each domain. The attachment of an SpAdomain to more than one site on a solid support, e.g., a chromatographyresin with epoxide or aldehyde group, can be achieved by reacting theamino group of lysine on SpA, via epoxide ring-opening or reductiveamination, respectively. In certain embodiments, multipoint attachmentcan be achieved by the reaction of one or more naturally occurring aminoacids on SpA having free hydroxyl groups, such as, for example, serineand tyrosine, with a support containing an epoxide group via aring-opening reaction. Alternatively, multipoint attachment can beachieved, for example, by the reaction of naturally occurring aminoacids on SpA having free carboxylic acid groups, such as, for example,aspartic acid and glutamic acid, with a support containing amino groupsvia, for example, N,N′-carbonyldiimidazole. Multipoint attachment of theligand to support can also be achieved by a combination of all the abovemechanisms.

To achieve caustic stability using the multimers of B and Z domains,this invention excludes the single cysteine mutation that leads tosingle point attachment.

SpA based chromatography ligands may also be attached to a solid supportvia an associative mechanism. For example, an associative group mayinteract with a ligand of interest non-covalently via ionic, hydrophobicor a combination of interactions, thereby to attach ligand of interestonto the solid surface. This facilitates the high efficiency coupling ofligand to the solid matrix, for example, as described in US PatentPublication No. 20070207500A1, thereby resulting in ligand densityhigher than that without the associative groups. Associative groupssuitable for use in the invention include charged species such as ionicspecies, and uncharged species such as hydrophobic species. Theassociative group may modify the solid support, e.g. by covalentlybinding directly with the solid support. Suitable examples of ionicspecies may include quaternary amines, tertiary amines, secondaryamines, primary amines, a sulfonic group, carboxylic acid, or anycombination thereof. Suitable examples of hydrophobic species mayinclude a phenyl group, a butyl group, a propyl group, or anycombination thereof. It is also contemplated that mixed mode species maybe used. The associative group may also interact with the proteinligand. Thus the interaction between the associative group and theprotein ligand may be comprised of a mixture of interactions, e.g. ionicand hydrophobic species.

The associative group may be covalently coupled to the solid support byreacting a functional group on the solid support with a functional groupon the associative group. Suitable functional groups include, but arenot limited to amines, hydroxyl, sulfhydryl, carboxyl, imine, aldehyde,ketone, alkene, alkyne, azo, nitrile, epoxide, cyanogens and activatedcarboxylic acid groups. As an example, agarose beads contain hydroxylgroups which may be reacted with the epoxide functionality of apositively charged associative group, such as glycidyl trimethylammoniumchloride. A skilled artisan will appreciate that a plurality ofassociative groups may be coupled to the solid support provided that atleast one bifunctional associative group is used. Thus associativegroups may be coupled in tandem to the solid support or they may beindividually coupled directly to the solid support.

In some embodiments, the present invention provides associative groupsand/or protein ligands which may be coupled to a solid support via anintervening linker. The linker may comprise at least one functionalgroup coupled to a linking moiety. The linking moiety may comprise anymolecule capable of being coupled to a functional group. For example,the linking moiety may include any of an alkyl, an alkenyl, or analkynyl group. The linking moiety may comprise a carbon chain rangingfrom 1 to 30 carbon atoms. In some embodiments the linker may becomprised of more than 30 carbon atoms. The linking moiety may compriseat least one hetero-atom such as nitrogen, oxygen and sulfur. Thelinking moiety may be comprised of a branched chain, an unbranched chainor a cyclic chain. The linking moiety may be substituted with two ormore functional groups.

Choosing the appropriate buffer conditions for coupling a protein ligandto a solid support is well within the capability of the skilled artisan.Suitable buffers include any non-amine containing buffer such ascarbonate, bicarbonate, phosphate and acetate buffers. When associativechemistry is used, salt concentration of the buffer will depend on theassociative group used. For example, the salt concentration may be inthe range of 5 nM-100 mM. Where a charged species is used, the saltconcentration may be at least 5 nM but less than 0.1M, at least 5 nM butless than 0.01M, at least 5 nM but less than 0.001M. In certainembodiments, the salt concentration may be 0.01M. Where a hydrophobicspecies is used a high salt concentration is usually desirable. Thus thesalt concentration may be greater than 0.001 M, greater than 0.01 M, orgreater than 0.1 M.

In some embodiments, when associative chemistry is used, the reaction isperformed at a temperature ranging from 0° C. to 99° C. In certainembodiments the reaction method is practiced at a temperature less than60° C., less than 40° C., less than 20° C., or less than 10° C. In someembodiments the method of the invention is practiced at a temperature ofabout 4° C. In other embodiments the method of the invention ispracticed at a temperature of 20° C.

VI. Methods for Assaying for Alkaline Stability of the Attached Ligands

The increased alkaline stability of the ligands subsequent to theirattachment to a support can be assayed by using well known techniques inthe art and those described herein. For example, in some embodiments,alkaline stability of multimers of SpA domains attached to a resin,e.g., B and Z domains of SpA attached to a chromatography resin, asdescribed herein, can be confirmed by treatment of the resin with 0.5 MNaOH. It is to be understood that an increased stability means that theinitial IgG binding capacity is retained during a longer period of timethan what can be achieved by the wild type SpA molecule. For example, incase of the present invention, after 100 cycles, each including a 15 mintreatment with 0.5 N NaOH, the percentage of retained capacity of theSpA ligands, e.g., those comprising multiple B or Z domains, is at least1.5 times more, 2.0 times more, 2.5 times more, or 3 times more thanthat of wild type SpA. In one embodiments, the alkaline stability of thebound ligand, as assayed by the retention of IgG binding capacity overtime, is measured as follows. The binding capacity, referred to as Qd50%, is measured by obtaining the volume of IgG loaded to a UV_(280 nm)of 50% of the initial IgG concentration. Qd 50% of the initial virginresin packed in a column is measured first. The resin is then exposed toabout 10 cycles of 15 min exposure of 0.5 N NaOH at 0.8 ml/min. Qd 50%is measured again. This process is repeated until the resin is exposedto a total of about 100 cycles of 0.5 N NaOH. Qd 50% is measured onelast time and the results from resins made from different ligands arecompared with the wildtype SpA.

In another assay, caustic or alkaline stability of the resins ismeasured by static soaking of resins of interest. By soaking a measuredamount of resin in 0.5N NaOH for 25 hrs with gentle rotation andmeasuring IgG binding capacity before and after the NaOH soaking, thealkaline stability by way of retention of binding capacity of the resincan be determined.

VII. Methods of Purifying a Target Molecule Using a ChromatographyLigand of the Invention

In some embodiments, the present invention provides a method ofpurifying a target molecule from a mixture using the alkaline stablechromatography ligands described herein. The target molecule may be anymolecule which is recognized by an alkaline stable chromatography ligandprovided herein, where the ligand is coupled to a solid support.Examples of target molecules include immunoglobulins. Theimmunoglobulins may be polyclonal antibodies or a monoclonal antibody ora functional fragment thereof. Functional fragments include any fragmentof an immunoglobulin comprising a variable region that still bindsspecifically to its antigen while at the same time retaining its abilityto specifically bind to a protein ligand coupled to a solid support.

In some embodiments, a method of isolating a target molecule of interestusing an alkaline stable chromatography ligand described herein includesthe steps of: (a) contacting a solid support including an attached SpAbased alkaline stable chromatography ligand with a mixture comprising amolecule of interest under conditions such that the target moleculespecifically binds to the ligand; and (b) altering the conditions suchthat the target molecule is no longer bound to the ligand, therebyisolating the target molecule.

In some embodiments, the altering step includes altering the pH, suchthe target molecule is no longer bound to the ligand. In a particularembodiment, the pH is altered in a manner such that it is more acidicthan the pH conditions in step (a). For example, in one embodiment, step(a) may be performed at a neutral pH, or a pH ranging from about 6 toabout 8 and step (b) may be performed at an acidic pH, e.g., a pHranging from about 1 to about 5.

In another embodiment, step (b) comprises altering the saltconcentration of the buffer in use, such that the target molecule is nolonger bound to the ligand. For example, in one embodiment, a high saltconcentration, e.g., >0.1 M, may be used in step (a) and a lower saltconcentration, e.g., <0.1M may be used in step (b). Conversely, in someembodiments, a low salt concentration, e.g., <0.1M may be used in step(a) and a high salt concentration may be used in step (b). In stillother embodiments both the pH and the salt concentration of the buffermay be altered between step (a) and step (b).

One skilled in the art can readily determine the conditions suitable forbinding a target molecule to a ligand, and thereby alter the conditionsto disrupt the binding of the molecule to the ligand.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Generation of an SpA B Domain Variant Having an n+1Mutation, where n Represents an Asparagine

In an exemplary experiment, a synthetic gene encoding the “B domain” ofprotein A is obtained from DNA 2.0 (Menlo Park, Calif.). The 5′ end ofthe gene includes a codon for an initiating methionine as well as sixhistidine codons at the 3′ end of the gene. The gene is provided invector pJ56:8620 from DNA 2.0. The parent vector pJ56 confers resistanceto ampicillin, kanamycin, chloramphenicol and gentamicin. Appropriaterestriction enzyme sites for the subsequent cloning of the gene intoexpression vectors are introduced into the gene at both 5′ and 3′ ends.A plasmid map of the vector is shown in FIG. 5.

Saturation mutagenesis is subsequently used to mutate glutamic acid atposition 24 to all other naturally occurring amino acids except cysteine(C), serine (S), alanine (A), glycine (g), asparagine (N), and glutamine(Q) by PCR-based methods using the Phusion High-Fidelity DNA polymerase(New England Biolabs, Ipswich, Mass.). Primers are purchased from IDTDNA (Coralville, Iowa), as 100 μM solution in Tris EDTA buffer. Themutagenic primers have the sequence CTGCCGAACCTGAACNN SGAACAACGCAACGG(SEQ ID NO: 41) where NNS represents the three bases encoding the aminoacid at position 24. PCR is performed in 50 μL reactions containingdNTPs (0.2 mM each), 125 ng of each primer, 50 ng of template plasmid,and 1 U of Phusion enzyme. PCR is carried out according to the schemeoutlined in Table III.

TABLE III Cycle description Temperature Time # of cycles Initialdenaturation 95° C. 30 seconds 1 cycle Denaturation 95° C. 30 secondsAnnealing 55° C. 60 seconds 18 cycles Extension 68° C. 6 minutes

PCR reactions are treated with the restriction enzyme DpnI (New EnglandBiolabs, Ipswich, Mass.) to reduce wild type background. To each 50 μLPCR reaction, about 14 of DpnI enzyme is added and the samples areincubated for about one hour at 37° C.

E. coli NEB5a competent cells (New England Biolabs, Ipswich, Mass.) aretransformed with 2 μL of the DpnI-treated PCR reaction. Cells are thawedon ice, and 2 μL of the PCR reaction is added to 25 μL of cells.Following about a 30 minute incubation on ice, cells are heat shockedfor 30 seconds at about 42° C. Cells are allowed to recover for about 5minutes on ice, and then 125 μL of SOC media (New England BioLabs) isadded. Cells are incubated for about one hour at 37° C., and then 100 μLare plated on LB plates (Northeast Laboratory Services, Winslow, Me.)containing 100 μg/mL ampicillin and grown overnight at about 37° C.Positive clones are identified by testing for the expression of theproteins in total cell lysates using SDS PAGE.

In order to obtain purified DNA, individual colonies are picked forovernight culture in LB containing 100 μg/mL ampicillin. DNA is purifiedusing spin mini-prep kits from Qiagen (Valencia, Calif.).

Mini-prepped DNA is sequenced to confirm the identity of each clone (MWGBiotech, Huntsville Ala.). The resulting plasmid is used to transform E.coli NEB5a competent cells as described above.

Following the identification of positive clones, 35 mls overnightcultures are grown in Terrific Broth with 100 μg/ml ampicillin and cellsare pelleted by centrifugation at 13,500 g for 10 minutes. Pellets areresuspended in 10 mls of 20 mM imidazole in PBS (phosphate bufferedsaline), lysed by sonication and centrifuged at 13,500 g for 30 minutesto pellet insoluble debris. Lysates are subsequently applied to 750 μlof Ni-NTA resin that is pre-equilibrated with 10 column volumes of 20 mMimidazole in PBS. After washing with 20 column volumes of 20 mMimidazole in PBS, samples are eluted from the resin with 200 mMimidazole in PBS. Purified protein is dialyzed overnight against PBSusing Pierce Slide-A-Lyzer 3.5 MWCO dialysis cassettes. Followingdialysis, total protein quantitation is accomplished using the PierceMicroBCA assay, and samples are stored at −30° C.

The amino acid and nucleic acid sequences for the wt his-tagged B domainand the various n+1 mutants are depicted in FIGS. 6 and 7.

Example 2 Assaying the Expressed Proteins for Alkaline Stability

Affinity purified wildtype and mutant SpA his-tagged constructsdescribed in Example 1 are diluted with MilliQ water or with NaOH to afinal concentration of IN and incubated for six hours at roomtemperature. Biorad Micro Biospin 6 gel filtration columns are used toneutralize and buffer exchange 50 μl of each sample into PBS. Totalprotein quantitation is accomplished using the Pierce MicroBCA assay andsamples are diluted to10 μg/ml in PBS for loading onto the ELISA plate.Approximately 200 μl (2 μg) of treated PrA is adsorbed to the wells ofan ELISA plate for 2-24 hours at 37° C. Plates are blocked in PierceSuperblock Blocking Buffer in PBS (Superblock-PBS) for about 2 hours atroom temperature. Approximately 200 μl of Sigma human gamma globulindiluted to 0.05 mg/ml in Superblock-PBS (10 μg) is added to each well ofthe plate and binding is allowed to proceed for about 1 hour at roomtemperature. Following three washes with PBS containing about 0.05%Tween20 (PBS-T), plates are incubated with 200 μl of a 1:10,000 dilutionof a chicken IgY-HRP conjugate raised against human IgG for about 1 hourat room temperature. After the final three washes with PBS-T, plates aredeveloped with 100 μl of Pierce 1-Step Slow TMB ELISA for 30 minutes atroom temperature. The reaction is stopped by the addition of 100 pl of1N HCL and primary IgG binding is quantitated as absorbance read at 450nm. All samples are assayed in triplicate and data is analyzed as thechange in primary IgG binding before and after caustic treatment.

The result of an exemplary experiment assaying for the alkalinestability of the various n+1 B domain mutants is summarized in the bargraph in FIG. 8. The various constructs are depicted on the X-axis andthe corresponding percent of IgG binding remaining after six hours ofexposure to 1M NaOH is plotted on the Y-axis As depicted in the bargraph in FIG. 8, several of the n+1 mutants having a bulky amino acid atposition 24 of the B domain exhibit enhanced caustic stability.

In another experiment, SpA ligands having two or more B domains or twoor more Z domains of SpA are assayed for alkaline stability using theassay described above. In yet another experiment, SpA ligands havingthree B domains (B3), four B domains (B4), five B domains (B5), six Bdomains (B6) or seven B domains (B7) are assayed for alkaline stabilityusing the assay described above.

In an exemplary experiment, the percent of residual IgG binding capacityof the ligands B3, B4 and B5, as assayed using the ELISA based methoddescribed herein is as follows. The B3 ligand retains about 79% of itsresidual IgG binding capacity following a six hour exposure to 1M NaOH;the B4 ligand retains about 86% of its residual IgG binding capacityfollowing a six hour exposure to 1M NaOH; and the B5 ligand retainsabout 83% of its residual IgG binding capacity following a six hourexposure to 1M NaOH. The wt SpA ligand (nPrA) only retains about 49% ofits residual IgG binding capacity following a six hour exposure to 1MNaOH.

Example 3 Attachment of Wildtype SpA and SpA Variants to a Solid Support

Subsequent to the generation of the various SpA variants andidentification of those which are caustic stable using one or moreassays known in the art and those described in Example 2, the SpAvariants are attached to a support, e.g., a solid support. In anexemplary experiment, agarose beads (Sepharose 4B) (GE Healthcare,Piscataway N.J.) are crosslinked using epichlorohydrin according to apreviously described method (Porath and Fornstedt, J. Chromatography,51:479 (1979)). The agarose beads are reacted with positively chargedassociative groups, e.g., cations, according to the following method: 50mL of beads are added to 40 g of 75% wt glycidyl trimethylammoniumchloride (GTMAC), 10 mL Milli-Q® water (Millipore Corp., Billerica,Mass.) and 1.67 g 50% wt sodium hydroxide. The reaction is shakenvigorously (>100 rpm) on a rotary shaker overnight at room temperature.The beads are then filtered and washed with three 100 mL volumes ofMilli-Q® water (Millipore Corp, Billerica, Mass.).

The beads (50 mL, filtered cake) are added to a jar containing 15 mLs of4.6M NaOH. The mixture is slurried and then 19.5 mL of butanedioldiglycidylether (BUDGE) is added. This mixture is shaken at 35° C. forabout 2 hours. The beads are then washed with 750 mL of Milli-Q® water(Millipore Corp, Billerica, Mass.) and equilibrated with 250 mL of 10 mMNaHCO₃.

Immediately following the BUDGE activation step, 10 mL of the filteredbead cake is added to 10 mL solution of 10 mM NaHCO₃ containing a 15 g/Lconcentration of wild type SpA or an SpA ligand according to the presentinvention. The mixture is capped in a glass vial and the vial is rotatedin a hybridizer at 37° C. for about 2 hours. After two hours, the beadsare washed with 30 mLs of Milli-Q® water (Millipore Corp, Billerica,Mass.). The filtered bead cake (10 mL) is added to a jar containing a 10mL solution comprised of 1 mL of thioglycerol and 9 mL of a buffersolution with 0.2 M NaHCO₃ and 0.5 M NaCl. The mixture is slurried androtated overnight at room temperature. The beads are then washed with 30mL of the following buffers: 0.1 M Tris Buffer (pH 8), 0.1 M Tris Bufferwith 0.15 M NaCl (pH 8), 50 mM Acetic Acid (pH 4.5), PBS (pH 7.4) with0.002% sodium azide.

Resin samples coupled with different SpA variants are labeled asfollows: nPrA for wild type SpA; Z3 for SpA ligand containing three Zdomains; E3 for SpA ligand containing three E domains; D3 for SpA ligandcontaining three D domains; A3 for SpA ligand containing three Adomains, C3 for SpA ligand containing three C domains; B3 for SpA ligandcontaining three B domains; B4 for SpA ligand containing four B domains;B5 for SpA ligand containing 5 B domains; B5NF and B7NF containing 5 and7 B domains, respectively, and additionally containing a G29A mutationat position 29. Following attachment to a resin, the various SpAvariants are assayed for alkaline stability.

Example 4 Resin IgG Binding Capacity (Qd 50%) Tests Before and AfterCaustic Cycling

In an exemplary experiment, the various SpA constructs according to theinvention are assayed for the IgG binding capacity following attachmentto a support. In one exemplary experiment, a standard method for testingresin/media dynamic capacity using commercial polyclonal IgG is used.Briefly, resin coupled with an SpA ligand according to the presentinvention is packed into an Omnifit column (6.6 mm×70 mm) in PBS, pH 7.4and the flow rate is set at 300 cm/hr. The packed column is equilibratedwith PBS for 10 column volumes (CVs). Polyclonal IgG (Sigma-Aldrich, 2mg/mL in PBS, pH 7.4) is loaded onto the column until UV_(280 nm)reaches more than 50% of the initial IgG concentration. After washingwith equilibration buffer, IgG is eluted with 0.1 M citric acid, pH 3.0.After each run, the media is sanitized using 6M Guanidininehydrochloride. Qd 50% is calculated based on the amount of IgG loadedwhen _(UV280 nm) reaches 50% of the initial IgG concentration.

After the initial dynamic capacity measurement, the media is exposed to10 cycles of 15 min, 0.5 N NaOH (flow rate 100 cm/hr) followed byanother IgG dynamic capacity measurement. The media is subsequentlycontacted with another 10 cycles of 15 min, 0.5N NaOH exposure, followedby another dynamic binding capacity measurement. Dynamic capacitymeasurement is carried out after each sample is exposed to 100 cycles of15 min 0.5 N NaOH.

In an exemplary experiment, the results of which are depicted in FIG. 9,trimers of E, D, A, B, C, and Z domains containing natural B domainlinkers are attached to an agarose resin and their caustic stability ismeasured by dynamic binding capacity over 100 cycles of causticexposure, each cycle including a 15 minute exposure to 0.5 N NaOH. Assummarized in the graph in FIG. 9, the number of cycles are plotted onthe X-axis and the IgG binding capacity is plotted on the Y-axis. The Cand the Z domain trimers exhibit approximately the same level of causticstability. The B domain trimers are more caustic stable than the Adomain trimers, which are more caustic stable than the E and the Ddomain trimers. Therefore, the order of caustic stability can besummarized as follows C and Z>B>A>E and D.

In another exemplary experiment, agarose resins coupled with B domainvariants: BBB (B3), BBBB (B4), BBBBB (B5), BBBBB-NF (B5-NF), andBBBBBBB-NF (B7-NF), are compared with wt SpA (nPrA) using the assaydescribed above to assay for IgG dynamic binding capacity over 100cycles of caustic exposure, each cycle including a 15 minute exposure to0.5 N NaOH. As summarized in the graph in FIG. 10, the extent of causticstability is directly proportional to the number of domains and furtherthat, the G29A mutation to reduce Fab binding does not affect thecaustic stability.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this inventionand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisinvention. All publications and inventions are incorporated by referencein their entirety. To the extent that the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supercede any such material. The citationof any references herein is not an admission that such references areprior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may vary depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. An alkaline-stable chromatography ligand comprising two or more Bdomains or two or more Z domains of Staphylococcus protein A (SpA), or afunctional fragment or variant thereof.
 2. An alkaline-stablechromatography ligand comprising two or more B domains or two or more Zdomains of Staphylococcus protein A (SpA), or a functional fragment orvariant thereof, wherein the two or more B domains or two or more Zdomains are attached to a chromatography resin at more than one site onthe resin.
 3. The alkaline-stable chromatography ligand of claim 1,wherein the ligand comprises three or more B domains or three or more Zdomains of SpA.
 4. The alkaline-stable chromatography ligand of claim 1,wherein the ligand comprises four or more B domains or four or more Zdomains of SpA.
 5. The alkaline-stable chromatography ligand of claim 1,wherein the ligand comprises five or more B domains or five or more Zdomains of SpA.
 6. The alkaline-stable chromatography ligand of claim 1,wherein the ligand comprises six or more B domains or six or more Zdomains of SpA.
 7. The alkaline-stable chromatography ligand of claim 1,wherein the ligand comprises seven or more B domains or seven or more Zdomains of SpA.
 8. The alkaline-stable chromatography ligand of claim 1,further comprising at least one amino acid mutation to alter Fabbinding.
 9. The alkaline-stable chromatography ligand of claim 2,wherein the ligand comprises three or more B domains or three or more Zdomains of SpA.
 10. The alkaline-stable chromatography ligand of claim2, wherein the ligand comprises four or more B domains or four or more Zdomains of SpA.
 11. The alkaline-stable chromatography ligand of claim2, wherein the ligand comprises five or more B domains or five or more Zdomains of SpA.
 12. The alkaline-stable chromatography ligand of claim2, wherein the ligand comprises six or more B domains or six or more Zdomains of SpA.
 13. The alkaline-stable chromatography ligand of claim2, wherein the ligand comprises seven or more B domains or seven or moreZ domains of SpA.
 14. The alkaline-stable chromatography ligand of claim2, further comprising at least one amino acid mutation to alter Fabbinding.
 15. The alkaline-stable chromatography ligand of claim 8,wherein the mutation comprises substitution of glycine at position 29with an alanine.
 16. A chromatography matrix comprising a ligandaccording to claim
 1. 17. A chromatography matrix comprising a ligandaccording to claim
 2. 18. A method of affinity purifying one or moreimmunoglobulins from a sample, the method comprising the steps of: (a)providing a sample comprising one or more immunoglobulins; (b)contacting the sample with the matrix of claim 16 under conditions suchthat the one or more immunoglobulins bind to the matrix; and (c)recovering the one or more bound immunoglobulins by eluting undersuitable conditions.
 19. A method of affinity purifying one or moreimmunoglobulins from a sample, the method comprising the steps of: (d)providing a sample comprising one or more immunoglobulins; (e)contacting the sample with the matrix of claim 17 under conditions suchthat the one or more immunoglobulins bind to the matrix; and (f)recovering the one or more bound immunoglobulins by eluting undersuitable conditions.
 20. The ligand according to claim 1, wherein theligand retains at least 95% of its binding capacity after 5 hours, orafter 10 hours, or after 15 hours, or after 20 hours incubation in 0.5 MNaOH.
 21. The ligand according to claim 2, wherein the ligand retains atleast 95% of its binding capacity after 5 hours, or after 10 hours, orafter 15 hours, or after 20 hours incubation in 0.5 M NaOH
 22. Theligand according to claim 1, wherein the ligand is capable of binding atleast 1.5 times, or 2 times, or 3 times, or more of IgG as compared towtSpA.
 23. The ligand according to claim 2, wherein the ligand iscapable of binding at least 1.5 times, or 2 times, or 3 times, or moreof IgG as compared to wtSpA.
 24. An alkaline-stable chromatographyligand comprising one or more isolated E, D, A, B, C or Z domains ofStaphylococcus protein A, wherein the one or more isolated domainscomprise one or more amino acid residues at position n+1 mutated to anynaturally occurring amino acid other than cysteine (C), serine (S),alanine (A), glycine (G), asparagine (N), or glutamine (Q), wherein nrepresents an asparagine.
 25. An alkaline-stable chromatography ligandcomprising one or more isolated E, D, A, B, C or Z domains ofStaphylococcus protein A, wherein the one or more isolated domainscomprise one or more amino acid residues at position n+1 mutated to anamino acid selected from the group consisting of arginine (R), asparticacid (D), glutamic acid (E), histidine (H), isoleucine (I), leucine (L),lysine (K), methionine (M), phenylalanine (F), proline (P), threonine(T), tryptophan (W), tyrosine (Y) and valine (V), wherein n representsan asparagine.
 26. The ligand of claim 24, wherein n is asparagine atposition
 23. 27. The ligand of claim 25, wherein n is asparagine atposition
 23. 28. A caustic stable affinity chromatography matrixcomprising three or more, or four or more, or five or more, or six ormore, or seven or more B or Z domains of SpA attached to a solid supportvia multipoint attachment.
 29. A method for identification of an SpAbased chromatography ligand which is caustic stable, the methodcomprising the steps of: (a) treating an SpA based ligand with caustic;(b) adding the caustic treated SpA ligand to a multiwell plate; (c)contacting the SpA based ligand with a saturating amount of a firstimmunoglobulin, such that the first immunoglobulin binds to the ligand;(d) contacting the bound first immunoglobulin with a molecule capable ofbinding the first immunoglobulin, wherein the molecule is conjugatedwith a detectable agent; and (e) detecting the amount of a signal fromthe agent, wherein the amount of signal is proportional to the causticstability of the ligand, thereby to identify the SpA based ligand asbeing caustic stable.
 30. The method of claim 29, wherein the moleculeis a second immunoglobulin.